Abstract

BACKGROUND:

Antigen-loaded dendritic cells (DC) are capable of priming naïve T cells and therefore represent an attractive adjuvant for vaccine development in anti-tumor immunotherapy. Numerous protocols have been described to date using different maturation cocktails and time periods for the induction of mature DC (mDC) in vitro. For clinical application, the use of mDC that can be generated in only three days saves on the costs of cytokines needed for large scale vaccine cell production and provides a method to produce cells within a standard work-week schedule in a GMP facility.

METHODS:

In this study, we addressed the properties of antigen uptake, processing and presentation by monocyte-derived DC prepared in three days (3d mDC) compared with conventional DC prepared in seven days (7d mDC), which represent the most common form of DC used for vaccines to date.

RESULTS:

Although they showed a reduced capacity for spontaneous antigen uptake, 3d mDC displayed higher capacity for stimulation of T cells after loading with an extended synthetic peptide that requires processing for MHC binding, indicating they were more efficient at antigen processing than 7d DC. We found, however, that 3d DC were less efficient at expressing protein after introduction of in vitro transcribed (ivt)RNA by electroporation, based on published procedures. This deficit was overcome by altering electroporation parameters, which led to improved protein expression and capacity for T cell stimulation using low amounts of ivtRNA.

CONCLUSIONS:

This new procedure allows 3d mDC to replace 7d mDC for use in DC-based vaccines that utilize long peptides, proteins or ivtRNA as sources of specific antigen.

Morphology and FITC-dextran uptake of 3d mDC and 7d mDC. The size and the morphology of immature and mature 3d DC and 7d DC were analyzed by (A) flow cytometry and (B) light microscopy. (C) DC were incubated without or with 10 μg/ml FITC-dextran at 37°C or at 4°C for 1 hour. The cells were then washed three times in ice-cold PBS with 1% FCS. The uptake of FITC-dextran was analyzed by flow cytometry. Data are representative for three independent experiments. The left-most open histograms represent medium only controls, the open grey curves indicate mDC incubated with FITC-Dextran at 4°C and the filled histograms at 37°C.

Phenotype of immature and mature DC. The expression of cell surface molecules on 2d iDC, 6d iDC, 3d mDC and 7d mDC was detected with specific antibodies and analyzed by flow cytometry. The open histograms correspond to the isotype controls, whereas the grey and black histograms display the specific binding of FITC- or PE-coupled antibodies. (A) Expression of CD14, CD83, CD209, CD40, HLA-DR and CCR7. (B) Expression of the B7-family-members CD86, CD80 and CD274. (C) Comparison of the expression of the costimulatory molecule CD80 and the inhibitory molecule CD274 on 3d mDC and 7d mDC. Data are representative for three independent experiments.

Migratory capacity of immature and mature DC. 2d iDC, 6d iDC, 3d mDC and 7d mDC were compared for their migratory capacity towards migration medium containing (+) or lacking (-) CCL19 in a trans-well migration assay. To measure directed migration, the medium in the lower chamber of the trans-well plate was supplied with 100 ng/ml CCL19, spontaneous migratory capacity was detected using medium that did not contain CCL19 in the lower chamber and random cell chemokinesis was determined by adding CCL19 to both the upper and the lower chambers. 2 × 105 DC were added in the upper chamber and incubated at 37°C and 5% CO2 for 2 h. Afterwards, DC numbers in the lower chambers were determined. Shown are three independent donors as mean values with standard errors of the mean (SEM). Statistical analyses were performed using the Mann Whitney test (n.s.: not significant).

Recognition of MART-1/Melan-A peptide on 3d mDC and 7d mDC by MART-1/Melan-A-specific CTL. (A) 3d mDC and 7d mDC were exogenously loaded with 10 μg/ml short MART-1/Melan-A26-35 peptide for 2 h or 24 h at 37°C and 5% CO2. After washing, the peptide-loaded DC were cocultured with MART-1/Melan-A-specific A42 CTL for 24 h at 37°C and 5% CO2. MART-1/Melan-A-positive tumor cells (Mel-93.04A12) and MART-1/Melan-A-negative tumor cells (Mel A375) served as controls and were cocultured with A42 CTL at the same time points as the DC (2 h and 24 h). The IFN-γ release of A42 CTL was measured by IFN-γ-ELISA. The columns show mean values of triplicates with standard deviations. Data are representative for two experiments. (B) 3d mDC and 7d mDC were incubated with different amounts of long MART-1/Melan-A peptide for 24 h at 37°C and 5% CO2. The DC were cocultured with A42 CTL for additional 24 h. The IFN-γ release of A42 CTL was measured by IFN-γ-ELISA. The columns show mean values of duplicates with standard deviations. The data for 2.5 μg/ml are representative for two independent experiments (n.d.: not detected).

Stimulation of naïve T cells with MART-1/Melan-A peptide-pulsed 3d and 7d mDC. Autologous PBL were stimulated with MART-1/Melan-A peptide-pulsed 3d and 7d mDC for 7 days, followed by specific restimulation for 24 h using peptide-pulsed mDC and Melan-A-positive tumor cells. The IFN-γ release of the PBL was measured by IFN-γ-ELISA. Shown are two independent donors as mean values with SEM (n.d.: not detected).